4, 7, 2 '-trichloro-7'-phenyl-6-carboxyl fluorescein as well as preparation method and application thereof

Abstract

The invention discloses 4, 7, 2 '-trichloro-7'-phenyl-6-carboxyl fluorescein as well as a preparation method and application thereof, and relates to the technical field of fluorescein synthesis. The preparation method comprises the following steps: reacting 6-carboxyl-tetrachloro-fluorescein, phenylboronic acid, a palladium catalyst and alkali in a mixed solvent in proportion at the temperature of 50-110 DEG C for 1.5-3 hours; 6-carboxyl-tetrachloro-fluorescein is used as a raw material, and separation and purification of ketone compound isomers are avoided; the synthesis route is short, operation is simple, and reaction conditions are mild; the use of dangerous chemical reagents is avoided; the reaction selectivity is good, byproducts are few, the product purity is high, and the total yield is high; the product can be amplified to a hectogram level, and batch production is easy to realize; unreacted raw materials in the reaction can be recycled, the purification frequency is low, and the process cost is remarkably reduced. And the prepared 4, 7, 2 '-trichloro-7'-phenyl-6-carboxyl fluorescein is expected to be popularized and applied in the fields of analysis and detection and biomedicine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fluorescein synthesis, in particular to 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein and a preparation method and application thereof. BACKGROUND

[0002] Fluorescein compounds are an important class of fluorescent dyes, which are widely used in biological labeling, fluorescent probes and molecular imaging fields. As a fluorescein derivative with a special structure, 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein shows important application value in biomedical research and analytical detection due to its excellent optical performance and chemical stability.

[0003] The traditional synthesis method mainly adopts a step-by-step synthesis strategy: first, 2,5-dichloro trimellitic anhydride and 4-chloro-resorcinol are used as starting materials to perform a Friedel-Crafts acylation reaction to generate a mixture of isomer ketone intermediates, which are purified by column chromatography, and then the single configuration ketone intermediate is subjected to a condensation reaction with 2,4-dihydroxybiphenyl in methane sulfonic acid at 135 DEG C high temperature to obtain the target product.

[0004] However, the Friedel-Crafts acylation reaction has many technical bottlenecks in the actual operation process, for example: the aluminum chloride catalyst used in the reaction has strong Lewis acidity, which is difficult to remove completely after the reaction and can adversely affect the subsequent purification and product performance; the reaction scale is limited, usually only in milligrams to grams, which is difficult to meet the needs of industrial production; the overall synthesis route is long, involves multiple separation and purification processes, the yield of each step is low, and the overall yield is only 20%; the reaction process will simultaneously produce two isomers with similar physical and chemical properties, which are difficult to distinguish by conventional separation methods and must be purified by multiple silica gel column chromatography, resulting in a significant increase in process cost and limiting its promotion in large-scale applications.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to provide a 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein and a preparation method and application thereof to improve or solve the above technical problems.

[0007] The present application is implemented as follows: In a first aspect, the present application provides a preparation method of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein, comprising the following steps: 6-Carboxy-tetrachlorofluorescein, phenylboronic acid, palladium catalyst and base were reacted in a mixed solvent in a certain proportion and reacted at a temperature of 50℃-110℃ for 1.5h-3h to obtain 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein. The amount of 6-carboxy-tetrachlorofluorescein used is >100g.

[0008] Secondly, embodiments of the present invention provide a 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared by the aforementioned preparation method, wherein the yield of the 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein is ≥36% and the purity is ≥98%.

[0009] Thirdly, embodiments of the present invention provide an application of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared by the aforementioned preparation method, or 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared by the aforementioned method, in the fields of analytical detection and biomedical research.

[0010] The present invention has the following beneficial effects: The 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein, its preparation method, and its applications provided in this invention have the following characteristics: It uses 6-carboxy-tetrachlorofluorescein (6-TET), a simple and readily available raw material, avoiding the difficulties in separating and purifying the 5,6-position isomers of ketone compounds; the synthetic route is short and simple to operate, with mild reaction conditions; it avoids the use of hazardous chemical reagents; the reaction has good selectivity, few byproducts, high product purity, and improved overall yield; it can be scaled up to the hundred-gram scale, easily achieving mass production; unreacted raw materials can be recovered and reused, reducing the number of purification steps and significantly lowering process costs. The prepared 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein is expected to be widely applied in the fields of analytical detection and biomedicine. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 The appearance diagram of the prepared 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein; Figure 2 The 1H NMR spectrum of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared in Example 1; Figure 3 ESI-MS analysis data of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared in Example 1; Figure 4 The data are HPLC analysis data of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared in Example 1. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0014] This invention provides a 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein that is readily available from simple and readily pure raw materials, has a short process route, is easy to operate, and is readily available for mass production. Specific implementation methods are as follows: In a first aspect, the present invention provides a method for preparing 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein, comprising the following steps: 6-Carboxy-tetrachlorofluorescein, phenylboronic acid, palladium catalyst and base were reacted in a mixed solvent in a certain proportion and reacted at a temperature of 50℃-110℃ for 1.5h-3h to obtain 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein. The amount of 6-carboxy-tetrachlorofluorescein used is >100g.

[0015] It should be noted that the preparation method provided in this invention is a palladium-catalyzed Suzuki coupling reaction. Compared with the existing Friedel-Crafts acylation reaction, its synthetic route is shorter and the reaction conditions are milder; it avoids the use of hazardous chemical reagents, eliminates the need to consider the difficult removal of aluminum trichloride catalyst and the need for multiple purifications of isomers; it has good reaction selectivity, can be scaled up to the hundred-gram scale, and the unreacted raw materials can be recovered and reused during the reaction, reducing the number of purification steps and significantly lowering the process cost. The reaction equation for the preparation of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein according to this invention is as follows: .

[0016] The existing Friedel-Crafts acylation reaction for the preparation of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein is shown in the following equation (Ref: WO1999016832A1): .

[0017] In an optional embodiment, the molar ratio of palladium catalyst to 6-carboxy-tetrachlorofluorescein is (0.1-0.3):1; The palladium catalyst is selected from at least one of palladium acetate, palladium chloride, tetra(triphenylphosphine)palladium, dichlorobis[di-tert-butyl(4-dimethylaminophenyl)phosphine]palladium(II) and bis(methyldiphenylphosphine)palladium(II) dichloride.

[0018] It should be noted that the present invention uses a palladium catalyst for palladium-catalyzed Suzuki coupling, which has relatively mild reaction conditions, readily available and low-cost raw materials, and high chemical selectivity. Through cross-coupling reaction, new groups can be attached at specific positions. For example, another functional group can be precisely attached to the halogen or borate ester position in the 6-carboxy-tetrachloro-fluorescein structure without affecting the properties of other parts of the molecule (such as the fluorophore).

[0019] For example, the molar ratio of palladium catalyst to 6-carboxy-tetrachlorofluorescein can be selected from any one of 0.1:1, 0.15:1, 0.2:1, 0.25:1 and 0.3:1, or other values ​​in (0.1-0.3):1.

[0020] The palladium catalyst remaining after the reaction can be recovered and reused.

[0021] In an optional embodiment, the molar ratio of the base to 6-carboxy-tetrachlorofluorescein is (1-5):1; The base is an inorganic base, selected from at least one of potassium carbonate, potassium phosphate, cesium carbonate, sodium carbonate, lithium carbonate, and sodium hydroxide.

[0022] The addition of alkali helps to neutralize the byproduct acid generated during the preparation of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein, ensuring that the reaction proceeds in the forward direction and maintaining the stability of the reaction system.

[0023] For example, the molar ratio of the base to 6-carboxy-tetrachloro-fluorescein can be selected from any one of 1:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 and 5:1, or other values ​​in (1-5):1.

[0024] In an optional embodiment, the mass ratio of the mixed solvent to 6-carboxy-tetrachlorofluorescein is (2-8):1; The mixed solvent includes an organic solvent and water in a volume ratio of 1:(0.2-5).

[0025] It should be noted that the use of organic solvent systems is beneficial for adjusting the polarity of the entire reaction system, providing a homogeneous environment for the reaction, improving reaction efficiency, and also facilitating the separation and purification of subsequent reaction products.

[0026] For example, the ratio of the mixed solvent to 6-carboxy-tetrachlorofluorescein can be selected from any one of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1, or other values ​​in the range of (2-8):1. The volume ratio of the organic solvent to water in the mixed solvent can be selected from any one of 1:0.2, 1:1, 1:2, 1:2.5, 1:3, 1:4, and 1:5, or other values ​​in the range of 1:(0.2-5).

[0027] In an optional embodiment, the organic solvent is selected from at least one of acetonitrile, dioxane, N,N-dimethylformamide, and tetrahydrofuran.

[0028] In an optional embodiment, the molar ratio of phenylboronic acid to 6-carboxy-tetrachlorofluorescein is (1-4):1.

[0029] Phenylboronic acid plays a crucial role in palladium-catalyzed coupling, functioning as a "carbon source donor + highly efficient metal transfer reagent." It combines high selectivity, functional group compatibility, and ease of operation, serving as a core building block for carbon-carbon bond construction. Through optimization of the base, ligand, solvent, and substrate structures, highly efficient and selective coupling reactions can be achieved, making it widely applicable in the synthesis of pharmaceuticals, materials, and natural products.

[0030] For example, the molar ratio of phenylboronic acid to 6-carboxy-tetrachlorofluorescein can be selected from any one of 1:1, 1.5:1, 2:1, 3:1, 3.5:1 and 4:1, or other values ​​in the range of (1-4):1.

[0031] In an optional embodiment, the preparation method further includes mixing the dissolved alkaline solution with the dissolved 6-carboxy-tetrachlorofluorescein solution, and after no bubbles are generated in the mixture, adding phenylboronic acid and an organic solvent, and purging with a protective gas. After reacting for 10-20 minutes, palladium catalyst is added, a protective gas is purged, and the mixture is refluxed for 1.5-3 hours before post-treatment to obtain 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein. The condensation reflux treatment temperature is 102℃-108℃.

[0032] It should be noted that after the reaction with palladium catalyst is completed, the reaction needs to be monitored by HPLC (high performance liquid chromatography). When the ratio of raw materials to products shows no significant change, the reaction solution is processed for post-processing.

[0033] In the embodiments of this invention, the HPLC test conditions are as follows: the chromatographic column used is a Waters XBridge C18, 4.6*150 mm, 3.5 μm, and the mobile phase is 0.1 M TEAA and water. In other embodiments of this invention, the column type and other parameters can be adjusted according to actual needs.

[0034] This invention does not impose any particular limitation on the type of protective gas; nitrogen, argon, helium, etc., can be selected according to actual needs.

[0035] The alkaline solution is prepared by dissolving the corresponding alkali in water; the 6-carboxy-tetrachloro-fluorescein solution is prepared by dissolving the raw material 6-carboxy-tetrachloro-fluorescein in water; in the process of preparing the above solutions, in order to accelerate the dissolution rate, appropriate heating or auxiliary stirring can be selected according to actual needs.

[0036] In an embodiment of the present invention, an alkaline solution and a 6-carboxy-tetrachloro-fluorescein solution are mixed and stirred until no more bubbles are generated in the solution before adding the next substance. Specifically, in an embodiment of the present invention, phenylboronic acid is added, and a protective gas is introduced to promote the full progress of the reaction and to protect the substances in the reaction process. After reacting for 10-20 minutes, a palladium catalyst is added, and a protective gas is continuously introduced. The reaction is carried out at a temperature of 50°C-110°C for 1.5-3 hours.

[0037] After the reaction is complete, the reaction solution is cooled to room temperature and then filtered. The filtration process is repeated multiple times to ensure maximum recovery of the reaction product. The reaction solution is then collected. The pH of the reaction solution is adjusted with concentrated hydrochloric acid until it reaches a pH of 1.0-1.5, at which point an orange-red solid precipitates. The product system is then filtered. The filter cake is redissolved in acetonitrile alkaline solution until the pH of the solution reaches a pH of 11.0-11.5, and then filtered. To accelerate the redissolution rate, ultrasonic treatment or stirring may be used as needed during the process.

[0038] Resolution with acetonitrile alkaline solution is beneficial for ensuring complete dissolution of the product in the acetonitrile alkaline solution, while avoiding side reactions and maintaining its reactivity and chemical stability.

[0039] Filtration after reconstitution is to remove insoluble solids from the reconstituted system.

[0040] In an optional implementation, the post-processing includes condensation reflux treatment, filtration treatment, pH adjustment, centrifugation treatment, purification treatment, and drying treatment; After purification and pH adjustment to 1.0-1.5, 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein was obtained. The drying temperature is 38℃-43℃.

[0041] Secondly, embodiments of the present invention provide a 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared by the aforementioned preparation method, wherein the yield of the 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein is ≥36% and the purity is ≥98%.

[0042] Thirdly, embodiments of the present invention provide an application of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared by the aforementioned preparation method, or 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared by the aforementioned method, in the fields of analytical detection and biomedical research.

[0043] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0044] Example 1 This embodiment provides a method for preparing 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein, comprising the following steps: Add 120 g (233 mmol) of 6-carboxy-tetrachlorofluorescein and 250 mL of pure water to a 2 L three-necked flask to prepare an aqueous solution of 6-carboxy-tetrachlorofluorescein; dissolve alkali (129 g (932 mmol)) in 175 mL of water, heat and stir at 35 °C to sonicate and dissolve K2CO3 to prepare a K2CO3 solution.

[0045] K₂CO₃ solution was added dropwise to an aqueous solution of 6-carboxy-tetrachlorofluorescein. After no bubbles were generated, phenylboronic acid (85.3 g, 700 mmol) and an organic solvent (dioxane, 75 mL) were added. After bubbling with nitrogen for 10 min, palladium catalyst (dichlorobis[di-tert-butyl(4-dimethylaminophenyl)phosphine]palladium(II), 25 g, 35.3 mmol) was added. Nitrogen gas was replaced by bubbling again, and the mixture was refluxed at 105 °C for 3 h. The reaction was monitored by HPLC. When the ratio of reactants to products showed no significant change, the reaction solution was processed for post-treatment.

[0046] The reaction solution was cooled to room temperature and filtered three times. Hydrochloric acid was added to the filtrate to adjust the pH to 1.3, resulting in the precipitation of a large amount of solid. The solid was redissolved in 2 mol / L NaOH solution containing 8% MeCN, with ultrasonic stirring until completely dissolved. The pH was then adjusted to 11.5, and the redissolved solution was filtered until no solid residue remained. The liquid was loaded onto a DAC 200 purification system and purified by column chromatography. The collected 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein solution was adjusted to pH 1.3 with concentrated hydrochloric acid to precipitate the product. After standing, centrifugation, filtration, and drying, 35 g of orange powdery solid 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein was obtained with an HPLC purity of 99.1%. The collected 6-carboxy-tetrachloro-fluorescein solution was adjusted to pH 1.3 with hydrochloric acid to precipitate the product. After standing, centrifugation, filtration, and drying, 28 g of pink powdery solid 6-carboxy-tetrachloro-fluorescein was obtained with an HPLC purity of 98.7%.

[0047] The cross-regions of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein and 6-carboxy-tetrachlorofluorescein obtained by column chromatography were also precipitated, allowed to stand, centrifuged, and then ultrasonically stirred in 2 mol / L NaOH solution containing 8% MeCN until completely dissolved. The pH was adjusted to 11.5, and the solution was filtered until no solids were found. The liquid was loaded onto a DAC 200 purification system and subjected to column chromatography again to obtain 12 g of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein with an HPLC purity of 98.6%. 7 g of 6-carboxy-tetrachlorofluorescein was obtained with an HPLC purity of 98.2%. The yield of the two column chromatography analyses was 36.4%, and the actual conversion rate was 51.3%. The relevant HPLC test conditions were as follows: the chromatographic column used was a Waters XBridge C18, 4.6*150 mm, 3.5 μm, and the mobile phase was 0.1 M TEAA and water.

[0048] 1 ¹H NMR: (600 MHz, d6-DMSO) δppm: 14.04 (b, 1H), 11.07 (s, 1H), 10.47 (s, 1H), 8.10 (s, 1H), 7.34–7.31 (m, 4H), 7.27–7.24 (m, 1H), 7.20 (s, 1H), 6.91 (s, 1H), 6.87 (s, 1H), 6.82 (s, 1H). (¹H NMR spectrum is shown below.) Figure 2 As shown.

[0049] ESI-MS analysis data such as Figure 3 As shown.

[0050] HPLC analysis data such as Figure 4 As shown, the peak at 3.997 min is 6-carboxy-tetrachlorofluorescein, and the peak at 4.455 min is 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein.

[0051] Example 2 This embodiment provides a method for preparing 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein, comprising the following steps: Add 120 g (233 mmol) of 6-carboxy-tetrachlorofluorescein and 250 mL of pure water to a 2 L three-necked flask to prepare an aqueous solution of 6-carboxy-tetrachlorofluorescein; dissolve alkali (K2CO3, 64.5 g, 466 mmol) in 175 mL of water, heat and stir at 35 °C to sonicate and dissolve K2CO3 to prepare a K2CO3 solution.

[0052] K₂CO₃ solution was added dropwise to an aqueous solution of 6-carboxy-tetrachlorofluorescein. After no bubbles were generated, phenylboronic acid (83.5 g, 700 mmol) and an organic solvent (dioxane, 75 mL) were added. After bubbling with nitrogen for 10 min, palladium catalyst (dichlorobis[di-tert-butyl(4-dimethylaminophenyl)phosphine]palladium(II), 25 g, 35.3 mmol) was added. Nitrogen gas was replaced by bubbling again, and the mixture was refluxed at 105 °C for 3 h. The reaction was monitored by HPLC. When the ratio of reactants to products showed no significant change, the reaction solution was processed for post-treatment.

[0053] The reaction solution was cooled to room temperature and filtered three times. Hydrochloric acid was added to the filtrate to adjust the pH to 1.3, resulting in the precipitation of a large amount of solid. The solid was redissolved in 2 mol / L NaOH solution containing 8% MeCN, with ultrasonic stirring until completely dissolved. The pH was then adjusted to 11.5, and the redissolved solution was filtered until no solid residue remained. The liquid was loaded onto a DAC 200 purification system and purified by column chromatography. The collected 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein solution was adjusted to pH 1.3 with concentrated hydrochloric acid to precipitate the product. After standing, centrifugation, filtration, and drying, 18 g of orange powdery solid 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein was obtained with an HPLC purity of 99.2%. The collected 6-carboxy-tetrachloro-fluorescein solution was adjusted to pH 1.3 with hydrochloric acid to precipitate the product. After standing, centrifugation, filtration, and drying, 41 g of pink powdery solid 6-carboxy-tetrachloro-fluorescein was obtained with an HPLC purity of 98.7%.

[0054] The cross-regions of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein and 6-carboxy-tetrachlorofluorescein obtained by column chromatography were also precipitated, allowed to stand, centrifuged, and then ultrasonically stirred in 2 mol / L NaOH solution containing 8% MeCN until completely dissolved. The pH was adjusted to 11.5, and the solution was filtered until no solids were found. The liquid was loaded onto a DAC 200 purification system and subjected to column chromatography again to obtain 4 g of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein with an HPLC purity of 98.2%. 20 g of 6-carboxy-tetrachlorofluorescein was obtained with an HPLC purity of 98.5%. The yield of the two column chromatography analyses was 17.0%, and the actual conversion rate was 34.8%. The HPLC test conditions were the same as in Example 1.

[0055] Example 3 This embodiment provides a method for preparing 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein, comprising the following steps: Add 120 g (233 mmol) of 6-carboxy-tetrachlorofluorescein and 250 mL of pure water to a 2 L three-necked flask to prepare an aqueous solution of 6-carboxy-tetrachlorofluorescein; dissolve alkali (K2CO3, 64.5 g, 466 mmol) in 100 mL of water, heat and stir at 35 °C to sonicate and dissolve K2CO3 to prepare a K2CO3 solution.

[0056] K₂CO₃ solution was added dropwise to an aqueous solution of 6-carboxy-tetrachlorofluorescein. After no bubbles were generated, phenylboronic acid (57.6 g, 460 mmol) and an organic solvent (dioxane, 75 mL) were added. After bubbling with nitrogen for 10 min, palladium catalyst (dichlorobis[di-tert-butyl(4-dimethylaminophenyl)phosphine]palladium(II), 25 g, 35.3 mmol) was added. Nitrogen gas was replaced by bubbling again, and the mixture was refluxed at 105 °C for 3 h. The reaction was monitored by HPLC. When the ratio of reactants to products showed no significant change, the reaction solution was processed for post-treatment.

[0057] The reaction solution was cooled to room temperature and filtered three times. Hydrochloric acid was added to the filtrate to adjust the pH to 1.3, resulting in the precipitation of a large amount of solid. The solid was redissolved in 2 mol / L NaOH solution containing 8% MeCN, with ultrasonic stirring until completely dissolved. The pH was then adjusted to 11.5, and the redissolved solution was filtered until no solid residue remained. The liquid was loaded onto a DAC 200 purification system and purified by column chromatography. The collected 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein solution was adjusted to pH 1.3 with concentrated hydrochloric acid to precipitate the product. After standing, centrifugation, filtration, and drying, 12 g of orange powdery solid 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein was obtained with an HPLC purity of 99.0%. The collected 6-carboxy-tetrachloro-fluorescein solution was adjusted to pH 1.3 with hydrochloric acid to precipitate the product. After standing, centrifugation, filtration, and drying, 48 g of pink powdery solid 6-carboxy-tetrachloro-fluorescein was obtained with an HPLC purity of 98.7%.

[0058] The cross-regions of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein and 6-carboxy-tetrachlorofluorescein obtained by column chromatography were also precipitated, allowed to stand, centrifuged, and then ultrasonically stirred in 2 mol / L NaOH solution containing 8% MeCN until completely dissolved. The pH was adjusted to 11.5, and the solution was filtered until no solids were found. The liquid was loaded onto a DAC 200 purification system and subjected to column chromatography again to obtain 3 g of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein with an HPLC purity of 98.4%. 24 g of 6-carboxy-tetrachlorofluorescein was obtained with an HPLC purity of 98.7%. The yield of the two column chromatography analyses was 11.6%, and the actual conversion rate was 29.1%. The HPLC test conditions were the same as in Example 1.

[0059] Test Example 1 This test case summarizes the yield, actual conversion rate and final purity of the products obtained in Examples 1-3 after two column chromatography analyses. The relevant data are shown in Table 1.

[0060] Table 1 Product Performance Data

[0061] In summary, the 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein, its preparation method, and its applications provided in this invention have the following characteristics: It uses 6-carboxy-tetrachlorofluorescein (6-TET), a simple and readily available raw material, avoiding the difficulties in separating and purifying the 5,6-position isomers of ketone compounds; the synthetic route is short and simple to operate, with mild reaction conditions; it avoids the use of hazardous chemical reagents; the reaction has good selectivity, few byproducts, high product purity, and improved overall yield; it can be scaled up to the 100-gram scale, easily achieving mass production; unreacted raw materials can be recovered and reused, reducing the number of purification steps and significantly lowering process costs. The prepared 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein is expected to be widely applied in the fields of analytical detection and biomedicine.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein, characterized in that, Includes the following steps: 6-Carboxy-tetrachlorofluorescein, phenylboronic acid, palladium catalyst and base were reacted in a mixed solvent in a certain proportion and reacted at a temperature of 50℃-110℃ for 1.5h-3h to obtain 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein. The amount of 6-carboxy-tetrachlorofluorescein used is >100g.

2. The preparation method according to claim 1, characterized in that, The molar ratio of the palladium catalyst to 6-carboxy-tetrachlorofluorescein is (0.1-0.3):1; The palladium catalyst is selected from at least one of palladium acetate, palladium chloride, tetra(triphenylphosphine)palladium, dichlorobis[di-tert-butyl(4-dimethylaminophenyl)phosphine]palladium(II) and bis(methyldiphenylphosphine)palladium(II).

3. The preparation method according to claim 1, characterized in that, The molar ratio of the base to 6-carboxy-tetrachlorofluorescein is (1-5):1; The alkali is an inorganic alkali, selected from at least one of potassium carbonate, potassium phosphate, cesium carbonate, sodium carbonate, lithium carbonate, and sodium hydroxide.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the mixed solvent to 6-carboxy-tetrachloro-fluorescein is (2-8):1; The mixed solvent comprises an organic solvent and water in a volume ratio of 1:(0.2-5).

5. The preparation method according to claim 4, characterized in that, The organic solvent is selected from at least one of acetonitrile, dioxane, N,N-dimethylformamide, and tetrahydrofuran.

6. The preparation method according to claim 1, characterized in that, The molar ratio of phenylboronic acid to 6-carboxy-tetrachlorofluorescein is (1-4):

1.

7. The preparation method according to claim 1, characterized in that, The preparation method further includes mixing the dissolved alkaline solution with the dissolved 6-carboxy-tetrachlorofluorescein solution, and after no bubbles are generated in the mixture, adding phenylboronic acid and organic solvent, and purging with a protective gas. After reacting for 10-20 minutes, palladium catalyst is added, a protective gas is purged, and the mixture is refluxed for 1.5-3 hours before post-treatment to obtain 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein. The condensation reflux treatment temperature is 102℃-108℃.

8. The preparation method according to claim 7, characterized in that, The post-processing includes filtration, pH adjustment, centrifugation, purification, and drying. After adjusting the pH of the reaction system to 1.0-1.5, the mixture was filtered and the filter cake was redissolved with acetonitrile alkaline solution until completely dissolved. After the pH of the solution reached 11.0-11.5, the mixture was purified. After purification and pH adjustment to 1.0-1.5, 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein was obtained. The drying temperature is 38℃-43℃.

9. A 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared by the method according to any one of claims 1-8, characterized in that, Yield of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein ≥36%, purity ≥98%.

10. The application of 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein prepared by the preparation method according to any one of claims 1-8 or the 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein according to claim 9 in the fields of analytical detection and biomedical fields.

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